TRPC Channel Reduces The OGD Neurons Damage By Regulating Ca²⁺ And MTOR Singnaleling Pathway

Hypoxic-ischemic encephalopathy(HIE) is the brain damage of fetalor neonatal which induced by asphyxia during perinatal．It characterized bypartial or total lack of oxygen，reduced cerebral blood flow．Neuron deathis the most direct and serious consequences of HIE，so reduce the death isthe most important strategy to study and solve the nervous systemdiseaseses．The transient receptor potential canonical (TRPC) is a subfamily ofnonselective cation channel permeable to Ca2+，which is present in manycell types，including neurons．It participate in the regulation of the Ca2+andbe regarded as the molecular basis of store operated calciumchannel．Although it participate in the progress of HIBD，but there is stillsome controversy of its function．General studies have found that TRPCcan increase the cellular damage which induced by HIBD though cation inthe cells，however，some studies revealed that TRPC play a protective rolein ischemia anoxic process．mTOR（mammaian target of rapamycin）is a kind of untypicalserine/threonine protein kinase，it is high-profile as the central of signaling pathways which regulate cell growth，proliferation and survival．Mainupstream signaling molecules of mTOR are various growth factors，Ca2+，amino acids，etc．Activated mTOR not only can active the hat dependentprotein translation though4E-BP1but also can facilitate the translation ofthe protein with p70S6K． The current research on mTOR signalingpathway were focuses on the process of formation and development oftumor，there is little report about its function in the HIBD，expecially withthe TRPC.So this study focuses on the TRPC channel，Ca2+，mTOR signaling todiscuss the function of TRPC channel and its mechanism in HIE．Methods1. Sprague Dawley (SD) rats hippocampal neurons and PC12cells wereculture in vitro，the cell mold of OGD has successfully established bytreated with Oxygen-Glucose Deprivation(OGD)．In order to exclude theaffects of TRPC to normal cells and the damage of the progress of OGD，sham-OGD group was set up．Then MNC were used to block the NMDAreceptor、AMPA receptor and Ca2+channel to exclude the effect of NMDAreceptor、 AMPA receptor and Ca2+channel． Here is the groups ofexperiment．a．Controlb．Sham-OGD+SKF96365(0，1，10，25μM)c．Sham-OGD+MNC+SKF96365(0，1，10，25μM)} d．OGD+SKF96365(0，1，10，25μM)e．OGD+MNC+SKF96365(0，1，10，25μM)}f．VehicleAfter block the TRPC，the following parameters were used to observethe effect of TRPC on OGD neurons and OGD PC12cell，to identifywhether the TRPC has protective effect on OGD neurons．a．The cell viability were measured by MTTb．The damage of cell membrane were measured by LDHc．Cell mortality were detected by trypan blue exclusiond．Cell apoptosis were examinated by Hochest33342or flow cytometry2．Block the TRPC channel to reveal the mechanism of TRPC protectiverolea．Laser confocal were used to detect the calcium ion concentrationb．Western blot were used to reveal the expression of mTOR、4E-BP1、p70S6K and its expression of phosphorylated．3. Data were analyzed by SPSS17.0software，comparisons betweengroups were tested by One-Way ANOVA analysis．Results1．In OGD cases, the damage degree of hippocampal neurons and PC12cells were positively correlated with the concentration of SKF96365．Therewere decrease of cell viability（p＜0.05）；increase of apoptosis andmobility（p＜0.05）． 2．After block the TRPC，there were obvious decrease of intracellularcalcium（p＜0.05）；decrease of mTOR、4E-BP1、p70S6K phosphorylation（p＜0.05）．ConclusionThe results reveal that TRPC have neuroprotection on cell OGD insult，and the underlying mechanism may be partly related that TRPC regulatesthe active of mTOR and downstream proteins such as4E-BP1、p70S6Kthrough Ca2+in hippocampal neurons and PC12cells.TRPC may be a molecular target for brain damage therapy，upregulatedthe expression of TRPC，may able to enhance the ability of neurons toresist damage, and it also provide a new way for further research of TRPCchannel functions in the nervous system damage．...